The insulin receptor tyrosine kinase, through its action on RAS and PI 3-kinase signaling elements, activates serine/threonine protein kinases and phosphatases which in turn mediate its effects on metabolic and cellular processes. We developed a powerful new method to screen cDNA expression libraries for potential downstream effectors of the lipid products of PI 3-kinase. Using this method, we discovered the protein GRP1, which binds PtdIns(3,4,5)P3 through its PH domain and catalyzes guanine nucleotide exchange of ARF proteins through its Sec7 domain. This domain structure of GRP1 suggests a second paradigm for insulin receptor signaling based on the activation of ARF proteins. These GTP binding proteins appear to regulate such cellular processes as membrane budding and actin assembly, suggesting the model: IR yields IRS yields PI 3-kinase yields PtdIns(3,4,5)P3 yields GRP1 yields ARF yields bioeffect. The studies proposed here will test this novel hypothesis. Cellular sites of recruitment of GRP1 by insulin will be defined by immuno-microscopic methods to determine which cellular membrane systems might be targets of GRP1/ARF actions. We shall test whether insulin enhances ARF activation (GTP-binding), as does overexpression of GRP1. Whether GRP1 is required for this insulin action will be determined by gene knockout as well as expression of dominant inhibitory constructs of GRP1. We shall employ our cDNA expression library screening technique using 3' phosphoinositides as probes to test whether other GRP-like proteins are expressed in insulin sensitive cells that may act as downstream signaling elements. We shall also define the unique structural elements within the PH domain of GRP1 and related proteins that confer unique specificity for high affinity binding of PtdIns(3,4,5)P3. Finally, we shall determine whether GRP-like proteins and ARF proteins are required for biological effects of insulin (e.g., glucose transport, glycogen synthesis, lipolysis, membrane ruffling, cell adhesion). The experiments proposed may define a new paradigm for insulin signaling that involves the GTP-binding ARF proteins, and reveal novel elements in this signaling pathway.